The Erythropoietin Receptor Stimulates Rapid Cycling and Formation of Larger Red Cells During Mouse and Human Erythropoiesis [preprint]

Erythroid terminal differentiation entails cell divisions that are coupled to progressive decreases in cell size. EpoR signaling is essential for the survival of erythroid precursors, but it is unclear whether it has other functions in these cells. Here we endowed mouse precursors that lack the EpoR with survival signaling, finding that this was sufficient to support their differentiation into enucleated red cells, but that the process was abnormal. Precursors underwent fewer and slower cell cycles and yet differentiated into smaller red cells. Surprisingly, EpoR further accelerated cycling of early erythroblasts, the fastest cycling cells in the bone marrow, while simultaneously increasing their cell size. EpoR-mediated formation of larger red cells was independent of the established pathway regulating red cell size by iron through Heme-regulated eIF2α kinase (HRI). We confirmed the effect of Epo on red cell size in human volunteers, whose mean corpuscular volume (MCV) increased following Epo administration. This increase persisted after Epo declined and was not the result of increased reticulocytes. Our work reveals a unique effect of EpoR signaling on the interaction between the cell cycle and cell growth. Further, it suggests new diagnostic interpretations for increased red cell volume, as reflecting high Epo and erythropoietic stress

Transcranial magnetic stimulation as biomarker of excitability in drug development: A randomized, double-blind, placebo-controlled, cross-over study

Aims: The purpose of this study was to investigate pharmacodynamic effects of drugs targeting cortical excitability using transcranial magnetic stimulation (TMS) combined with electromyography (EMG) and electroencephalography (EEG) in healthy subjects, to further develop TMS outcomes as biomarkers for proof-of-mechanism in early-phase clinical drug development. Antiepileptic drugs presumably modulate cortical excitability. Therefore, we studied effects of levetiracetam, valproic acid and lorazepam on cortical excitability in a double-blind, placebo-controlled, 4-way cross-over study. Methods: In 16 healthy male subjects, single- and paired-pulse TMS-EMG–EEG measurements were performed predose and 1.5, 7 and 24 hours postdose. Treatment effects on motor-evoked potential, short and long intracortical inhibition and TMS-evoked potential amplitudes, were analysed using a mixed model ANCOVA and cluster-based permutation analysis. Results: We show that motor-evoked potential amplitudes decreased after administration of levetiracetam (estimated difference [ED] −378.4 μV; 95%CI: −644.3, −112.5 μV; P <.01), valproic acid (ED −268.8 μV; 95%CI: −532.9, −4.6 μV; P =.047) and lorazepam (ED −330.7 μV; 95%CI: −595.6, −65.8 μV; P =.02) when compared with placebo. Long intracortical inhibition was enhanced by levetiracetam (ED −60.3%; 95%CI: −87.1%, −33.5%; P <.001) and lorazepam (ED −68.2%; 95%CI: −94.7%, −41.7%; P <.001) at a 50-ms interstimulus interval. Levetiracetam increased TMS-evoked potential component N45 (P =.004) in a central cluster and decreased N100 (P <.001) in a contralateral cluster. Conclusion: This study shows that levetiracetam, valproic acid and lorazepam decrease cortical excitability, which can be detected using TMS-EMG–EEG in healthy subjects. These findings provide support for the use of TMS excitability measures as biomarkers to demonstrate pharmacodynamic effects of drugs that influence cortical excitability

Effects of erythropoietin on cycling performance of well trained cyclists: a double-blind, randomised, placebo-controlled trial

Background Substances that potentially enhance performance (eg, recombinant human erythropoietin [rHuEPO]) are considered doping and are therefore forbidden in sports; however, the scientific evidence behind doping is frequently weak. We aimed to determine the effects of rHuEPO treatment in well trained cyclists on maximal, submaximal, and race performance and on safety, and to present a model clinical study for doping research on other substances. Methods We did this double-blind, randomised, placebo-controlled trial at the Centre for Human Drug Research in Leiden (Netherlands). We enrolled healthy, well trained but non-professional male cyclists aged 18–50 years and randomly allocated (1:1) them to receive abdominal subcutaneous injections of rHuEPO (epoetin β mean dose 6000 IU per week) or placebo (0·9% NaCl) for 8 weeks. Randomisation was stratified by age groups (18–34 years and 35–50 years), with a code generated by a statistician who was not masked to the study. The primary outcome was exercise performance, measured as maximal power output (Pmax), maximal oxygen consumption VO2 max, and gross efficiency in maximal exercise tests with 25 W increments per 5 min, as lactate threshold and ventilatory threshold 1 (VT1) and 2 (VT2) at submaximal levels during the maximal exercise test, and as mean power, VO2, and heart rate in the submaximal exercise tests at the highest mean power output for 45 min in a laboratory setting and in a race to the Mont Ventoux (France) summit, using intention-to-treat analyses. The trial is registered with the Dutch Trial Registry (Nederlands Trial Register), number NTR5643. Findings Between March 7, 2016, and April 13, 2016, we randomly assigned 48 participants to the rHuEPO group (n=24) or the placebo group (n=24). Mean haemoglobin concentration (9·6 mmol/L vs 9·0 mmol/L [estimated difference 0·6, 95% CI 0·4 to 0·8]) and maximal power output (351·55 W vs 341·23 W [10·32, 3·47 to 17·17]), and VO2 max (60·121 mL/min per kg vs 57·415 mL/min per kg [2·707, 0·911 to 4·503]) in a maximal exercise test were higher in the rHuEPO group compared with the placebo group. Submaximal exercise test parameters mean power output (283·18 W vs 277·28 W [5·90, −0·87 to 12·67]) and VO2 (50·288 mL/min per kg vs 49·642 mL/min per kg [0·646, −1·307 to 2·600]) at day 46, and Mont Ventoux race times (1 h 40 min 32 s vs 1 h 40 min 15 s [0·3%, −8·3 to 9·6]) did not differ between groups. All adverse events were grade 1–2 and were similar between both groups. No events of grade 3 or worse were observed. Interpretation Although rHuEPO treatment improved a laboratory test of maximal exercise, the more clinically relevant submaximal exercise test performance and road race performance were not affected. This study shows that clinical studies with doping substances can be done adequately and safely and are relevant in determining effects of alleged performance-enhancing drugs. Funding Centre for Human Drug Research, Leiden